Mammalian spermatozoa have been known to be antigenic for many years. More recently, it has been demonstrated that mammalian sperm contain an antigenic enzyme, which is known as the C.sub.4 isozyme of lactate dehydrogenase (LDH-C.sub.4). LDH-C.sub.4 has been isolated in pure crystalline form from mouse testes. Goldberg (1972), J. Biol. Chem., 2247:2044-2048. The enzyme has a molecular weight of about 140,000 and is composed of four identical C subunits. The amino acid sequence and three-dimensional structure of mouse LDH-C.sub.4 have been studied and described by a number of investigators: Musick, et al., (1976), J. Mol. Biol., 104:659-668; Wheat, et al. (1977), Biochem. & Biophys. Res. Comm. 74, No. 3:1066-1077; Li, et al. (1983), J. Biol. Chem. 258:7017-7028; and Pan, et al. (1983), J. Biol. Chem. 258:7005-7016.
In 1974, Dr. Erwin Goldberg reviewed the effects of immunization with LDH-X (LDH-C.sub.4) on fertility, and advanced the possibility that "by using a defined macro-molecular constituent of sperm it becomes possible to elucidate its primary structure in terms of amino acid sequence, to map specifically the antigenic determinant(s) responsible for inducing infertility, and then to construct synthetic peptides containing these determinants. Possessing the capability for synthesizing a molecule with such properties makes the immunological approach to fertility control feasible". Karolinska Symposia on Research Methods in Reproductive Endocrinology 7th Symposium: Immunological Approaches to Fertility Control, Geneva, 1974, 202-222.
Subsequent investigations by Dr. Goldberg and his research associates identified several amino acid sequences of mouse LDH-C.sub.4 which in synthetic form (e.g., as short chain polypeptides) bind to LDH-C.sub.4 antiserum. See Wheat, et al. (1981), in Rich, et al., Peptides: Synthesis-Structure-Function, Proc. 7th Amer. Peptide Symp., pp. 557-560; and Gonzales-Prevatt, et al. (1982), Mol. Immunol. 19:1579-1585. Several antigenic peptide compounds based on these sequences have been patented. See U.S. Pat. Nos. 4,290,944; 4,310,456; 4,353,822; 4,377,516; 4,392,997; 4,278,519; and 4,585,587.
The antigenic peptides based on sequences of mouse LDH-C.sub.4 are potentially useful in preparing vaccines to reduce female fertility. Immunization of female mammals results in the development of circulating antibodies specific to LDH-C.sub.4. These immunoglobulins reach the female reproductive tract as a transudate of serum: Kille, et al. (1977), Biol. Reprod. 20:863-871. Antibody in cervical mucus, uterine fluids, and oviducal fluids combine with LDH-C.sub.4 on the sperm surface and impede the progress of the male gamete, presumably by agglutination. Systemic immunization with LDH-C.sub.4 markedly interferes with sperm transport in the female reproductive tract: Kille et al. (1980), J. Reprod. Immunol., 2:15-21.
The 1983 status of research on LDH-C.sub.4 and antigenic peptides for use in female contraceptive vaccines was summarized in two publications by the Goldberg group: Goldberg, et al. (1983), In Immunology of Reproduction, Chapt. 22, pp. 493-504; and Wheat, et al. (1983), in Isozymes: Current Topics in Biological and Medical Research, Vol. 7, pp. 113-140. The synthetic peptide corresponding to the mouse LDH-C.sub.4 sequence 5 to 15 (which included 16 amino acids because of Glu-14a and Asp-14b) was selected as a promising peptide for immunization studies. Wheat, et al. (1985), Molec. Immun. 22:1195-1199; and Goldberg and Shelton, in "Immunological Approaches to Contraception and Promotion of Fertility", pages 219-230 (ed. G. P. Talwar, Plenum Publishing Corp., 1986) and U.S. Pat. No. 4,392,997. With the peptide identified as mouse (MC) 5-15 immunization studies were conducted with female baboons. The results obtained indicated a 71% reduction in fertility, but in commenting thereon, Goldberg and Shelton stated (at page 225):
"While these results demonstrate the promise that this approach holds, the percentage of fertility reduction must be increased to a level acceptable for human contraceptive practice. Certainly, one possibility is that, while MC5-15 immunization is effective in reducing fertility, another peptide or combination of peptides might be more immunogenic."
One approach pursued by Dr. Goldberg and his colleagues to improve antifertility immunogenicity of the proposed vaccines was to determine the sequences of human LDH-C.sub.4. The human gene for Ldh-c was isolated, cloned, and sequenced: Millan, et al. (1987), Proc. Natl. Acad. Sci. USA, 84: 5311-5319. This led to the discovery that the human sequences of the LDH-C.sub.4 enzyme corresponding to the previously-identified antigenic sequences of mouse LDH-C.sub.4 differed markedly with respect to amino acid content. The comparative sequences are illustrated in Millan, et al., cited above, at page 5314, and in Goldberg and Millan U.S. Pat. No. 4,782,136. The 16 number sequence of the human enzyme corresponding to the mouse sequence 5-15 differed by six amino acids, as illustrated in column 3 of the cited Goldberg and Millan patent.